Non-isotopic detection of osteoplastic activity in vivo using modified bisphosphonates

ABSTRACT

The present invention is directed to a non-isotopic methods for the in vitro and in vivo detection of hydroxyapatite-positive cells and structures.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/424,572, filed Apr. 25, 2003, which is a continuation ofInternational Application No. PCT/US01/51312, which designated theUnited States and was filed Oct. 29, 2001, published in English, whichclaims the benefit of U.S. Provisional Application No. 60/244,020, filedOct. 27, 2000.

The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The development and maintenance of the vertebral skeleton is a complexprocess, but in simplest terms represents a balance betweenosteoblast-induced mineralization and osteoclast-induceddemineralization. Osteoblast-like cells are also present in the vascularwall and participate in the earliest manifestations of atherosclerosis.Hydroxyapatite (HA; Ca₁₀(PO₄)₆(OH)₂, also know as hydroxylapatite) isthe major mineral product of osteoblasts and calcifying vascular cellsand binds naturally occurring pyrophosphates and phosphonates with highaffinity. Osteoblastic activity occurs at sites of new bone formation,i.e., sites of deposition of hydroxyapatite. New bone formation occursafter fracture, at sites of bony infections, and especially at the sitesof certain cancer metastases (e.g., prostate cancer metastases).

At the present time, osteoblastic activity is detected in vivo usingradionuclides and SPECT imaging. For instance, a common technique is the“bone scan”, which utilizes the radiometal ^(99m)Tc coupled to thebisphosphonate compound methylene bisphosphonate (MDP). Unfortunately,radioscintigraphic detection does not provide high-resolution anatomicaldetail, and requires the use of radioactive compounds.

Fluorescence imaging is found at the heart of numerous chemical andbiomedical analysis schemes. Many of these schemes are based on theintroduction of a fluorescent species as a marker, stain, dye orindicator (Devoisselle et al. (1993) Optical Engineering 32:239;Haugland and Minta, “Design and Application of Indicator Dyes,”Noninvasive Techniques in Cell Biol., ed. B. H. Satir, Chap, 1, p 1,(Wiley-Liss, New York, N.Y., 1990); Gross, “Quantitative Single CellFluorescence Imaging of Indicator Dyes,” Noninvasive Techniques in CellBiol., ed. B. H. Satir, Chap. 2, p 21, (Wiley-Liss, New York, N.Y.,1990). To date, however, there has not been a non-isotopic method fordirectly detecting HA in vivo.

It is an object of the present invention to use bisphosphonate compoundsfor use in non-isotopic (i.e., without the need for radioactivity)detection of hydroxyapatite, such as to determine osteoblastic activityin vivo.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a contrast agentrepresented in the general formula [I] or pharmaceutically acceptablesalts thereof:

wherein

D represents a fluorescent moiety,

R represents a linking group that covalently links the dye (D) andbisphosphonate moiety;

R₁ represents H, —OH, or a halogen; and

R₂ represents, independently for each occurrence, a free electron pair,hydrogen, or a pharmaceutically acceptable counterion.

In certain embodiments, R is an amine substituted lower-alkyl whichforms an amide bond with a pendant group of the fluorescent moiety,e.g., such as —(CH₂)_(L)—NH—, where L is an integer from 1 to 6.

In certain embodiments, R′ is —OH or —Cl.

In certain embodiments, D is a near-infrared fluorescent moiety., e.g.,a near-infrared fluorescent dye. In certain embodiments, D is apolysulfonated indocyanine dye.

In certain embodiments, the fluorescent moiety is represented by theformula [II]:

wherein

L₁-L₇ are each, independently, a substituted or unsubstituted methine,provided that one of L₁-L₇ is substituted with the linker group R whichis attached to the bisphosphonate;

R₃, independently for each occurrence, is a substituted or unsubstitutedalkyl;

A and B are each, independently, 5-7 membered substituted orunsubstituted aromatic rings;

X and Y are the same or different and each is a group of the formula—O—, —S—, —CH═CH— or —C(R₄)₂—;

R₄, independently for each occurrence, is a hydrogen or substituted orunsubstituted lower alkyl; and

r is 0, 1 or 2

For example, fluorescent moiety [II] can have one or more of thefollowing features: (a) is free of a carboxylic acid group in amolecule; (b) r is 1; (c) includes 4 or more sulfonic acid groups; (d)includes 10 or less sulfonic acid groups; (e) A and B are,independently, benzo or naphtho rings; and (f) X and Y are —C(CH₃)₂—.

In certain embodiments, the fluorescent moiety is represented by theformula [III]

wherein

X and Y are the same or different and each is a group of the formula—C—, —S—, —CH═CH— or —C(R₄)₂—;

R₄, independently for each occurrence, is a hydrogen or substituted orunsubstituted lower alkyl; and

R₆, independently for each occurrence, is hydrogen or —SO₃R₇;

R₇, independently for each occurrence, is hydrogen or a pharmaceuticallyacceptable counter ion;

r is 0, 1, 2, 3, 4 or 5.

In many preferred embodiments, the fluorescent moiety is a near-infraredfluorescent moiety and has an extinction coefficient of at least 50,000M⁻¹ cm⁻¹ in aqueous medium, and even more preferably at least 100,000,200,000 or even 300,000 M⁻¹ cm⁻¹.

In many preferred embodiments, the fluorescent moiety is a near-infraredfluorescent moiety and has a quantum efficiency, Φ_(F), of at least 25%,and even more preferably at least 30%, or even 40%.

In preferred embodiments, the contrast agent has an LD₅₀ of 50 mg/Kg orgreater humans, and even more preferably of at least 100, 250 or even500 mg/Kg.

In preferred embodiments, the contrast agent has a half-life in thehuman body of at least 10 minutes, and even more preferably at least 20,30 or even 45 minutes.

Another aspect of the invention provides a contrast agent comprising abisphosphonate covalently linked to a near-infrared fluorescent moiety,wherein the contrast agent (a) has an extinction coefficient of at least100,000 M⁻¹ cm⁻¹ in aqueous medium, (b) has an LD₅₀ of at least 100mg/Kg in humans, and (c) has a half-life in the human body of at least10 minutes.

In certain embodiments, the bisphosphonate is selected from the groupconsisting of alendronate, clodronate, EB-1053, etidronate, ibandronate,incadronate, neridronate, olpadronate, phosphonate, palmidronate,risedronate, tiludronate, YH 529 and zoledronate.

Yet another aspect of the present invention provides methods formanufacturing a composition of for in vivo imaging by formulating acontrast agent described herein in a pharmaceutically acceptableexcipient.

Still another aspect of the invention provides a kit for in vivo imagingcomprising a contrast agent of the invention in association withinstructions for administering the contrast agent to a patient.

Another aspect of the invention provides a method for in vivo imaging oftissue with exposed hydroxyapatite, comprising,

(i) administering to the animal a contrast agent of any of claims 1-16in an amount sufficient to render hydroxyapatite-containing tissuedetectable by a fluorescent detector;

(ii) obtaining a fluorescent image of the animal, or at least a portionthereof, at a wave length(s) which detects the contrast agent;

(iii) constructing an image of the animal including the pattern ofdistribution of the contrast agent.

The subject method can be used for evaluating a bone for its conditionand/or biomechanical property, e.g., for determining bone matrix densityand/or detecting changes in bone matrix volume. For instance, the methodcan be used as part of a protocol for diagnosing osteoporosis. In otherembodiments, the method can be used for determining at least one ofanisotropic elastic constants, bone strength, or fracture risk.

The subject method can also be used for diagnostic detection of bonediseases accompanied with abnormality of calcium hydroxyapatite, such asfor detecting the presence of osteoblastic metastase.

Another embodiment of the method can be used for detecting of vesselmicro-calcification, such in the detection of sub-clinical vesselcalcification, e.g., as a way of more accurately assigningcardiovascular risk, as well as an adjunct to standard catheterizationprocedures, or as a prognostic tool to assess cardiovascular risk.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Conjugation of Pamidronate to IRDye78, Purification of theProduct and Spectral Characteristics:

A. The primary amine of pamidronate disodium (I) was conjugated to thesodium salt of the N-hydroxysuccinimide (NHS) ester of IRDye78 (II), asdescribed in the Experimental Protocol, yielding the NIR fluorescentproduct pamidronate/IRDye78 (III; Pam78). The chemical structure andmolecular weight (M.W.) for each compound at pH 7.4 is shown. Thecarboxylic acid of IRDye78 is compound I with a hydrogen replacing thesuccinimide group.

B. The reactants and products of the chemical reaction described in FIG.1A were separated using thin-layer chromatography (TLC) as described inthe Experimental Protocol. An actual TLC plate representative of thisprocedure is shown. The carboxylic acid of IRDye78 (Lane 1; arrowhead),free pamidronate (Lane 2; closed arrow; visualized with ninhydrin asdescribed in the Experimental Protocol) and Pam78 (Lane 3; open arrow)separated with R_(f) values of 0.8, 0, and 0.6, respectively. The originis indicated, and the mobile phase front is shown across the top of theplate as a dashed line.

C. Absorbance wavelength scans using IRDye79 (left panel) and Pam78(right panel) at a concentration of 1 μM in NK buffer. The wavelength ofpeak absorption is indicated.

D. Excitation//emission fluorescence wavelength scans using IRDye78(left panel) and Pam78 (right panel) at a concentration of 500 nM in NKbuffer. For fluorescence excitation scans (solid lines), emissionwavelength was fixed at 796 nm. For fluorescence emission scans (dashedlines), excitation wavelength was fixed at 771 nm. The peak wavelengthfor each scan is shown above the corresponding curve.

FIG. 2: Hydroxyapatite Binding Properties of IRDye78 and Pam78:

A. The kinetics of binding to hydroxyapatite (HA) crystals was measuredfor Pam78 (solid circles) and the carboxylic acid of IRDye78 (IRDye78alone; open circles), as described in the Experimental Protocol.Measurements were acquired at 5, 15, and 30 minutes, and 1, 2, 3, and 24hours after mixing. On the ordinate is shown the number of μmol of eachcompound bound per gram of HA (mean±SEM).

B. Steady state binding to HA crystals was measured for Pam78 (solidbars) and the carboxylic acid of IRDye78 (IRDye78 alone; open bars), asdescribed in the Experimental Protocol. On the abscissa is theconcentration of applied compound. On the ordinate is shown the numberof μmol of each compound bound per gram of HA (mean±SEM) at 3 hours.

C. Competition of Pam78 binding to HA using unlabeled pamidronate wasperformed as described in the Experimental Protocol. On the ordinate isshown the number of μmol of each compound bound per gram of HA(mean±SEM) relative to binding of Pam78 in the absence of unlabeledpamidronate.

D. Direct visualization of Pam78 binding to HA was accomplished usingNIR fluorescence microscopy as described in the Experimental Protocol.Phase contrast (left panels) and NIR fluorescence images (right panels)of HA crystals treated with 1 μM Pam78 (top panels) or the carboxylicacid of IRDye78 (bottom panels) are shown.

FIG. 3 In Vivo Biodistribution and Pharmacokinetics of IRDye78 and Pam78using Near-Infrared Fluorescence Imaging:

A. 2.6 nmol of IRDye78 carboxylic acid in 80 μl of PBS was injectedintravenously in the tail vein of a hairless (nu/nu) mouse and imagedwith the small animal imaging system as described in the ExperimentalProtocol. The left photographs show dorsal images and the rightphotographs show ventral images of the same mouse. For orientation, awhite light image and calibration bar are shown in the top leftphotograph. All NIR fluorescence images were acquired using a 500 msecexposure time and are normalized to the 15 minute image. The Time=0photograph shows NIR autofluorescence prior to compound injection.Dorsal and ventral images at 15 minutes and six hours post-injection areshown. In the 15 minute dorsal image, note is made of sub-millimeterskin defects (D) which appear highlighted after injection. In the 15minute ventral image, note is made of intense signal in the bladder (B).In the six hour ventral image, note is made of signal emanating fromloops of the large (LI) and small (SI).

B. 2.6 nmol of Pam78 in 80 μl of PBS was injected intravenously in thetail vein of a hairless (nu/nu) mouse and dorsal images were obtainedwith the small animal imaging system described in the ExperimentalProtocol. For orientation, a white light image and calibration bar areshown in the top left photograph. All NIR fluorescence images wereacquired using a 500 msec exposure time and are normalized to the sixhour image of FIG. 4A (left panel). The Time=0 image shows NIRautofluorescence prior to compound injection. As the injectedfluorophore is cleared, areas of high HA in the skeleton become visible.As early as 15 minutes after injection, ribs (R), spine (Sp) and arevisualized. Signal emanating from the kidneys is also indicated (K). By3 hours, the phalanges (P), wrist (W), skull (Sk), pelvis (Pe), femur(F) and ankle (A) become visible.

C. Serum concentration of IRDye78 carboxylic acid (left panel; opencircles) and Pam78 (right panel; closed circles) were measured asdetailed in the Experimental Protocol after intravenous injection of 2.6nmol of each compound into the tail vein. Ordinate shows serumconcentration as measured spectrophotometrically. Abscissa shows time inminutes.

FIG. 4: High Resolution In Vivo Near-Infrared Fluorescence Imaging ofHA: Specific anatomic sites of the same living animal shown in FIGS. 3Band 4A (left panel) were imaged six hours post injection of Pam78 usingthe zoom lens capability of the small animal imaging system described inthe Experimental Protocol. Images were normalized for optimal appearanceof each bony structure. For orientation, calibration bars are shown ineach photograph:

A. Ventral image of the head showing intense staining of the nasal bone(Na) and maxilla (Mx). The anterior palatine foramen (APF) is alsovisible, as is the (out of focus) mandible (Ma).

B. Oblique image of rib cage showing costochrondral junction (arrow,CCJ).

C. The thoracic spine showing spinous (SP) and transverse processes(TP).

D. The front paw showing individual phalanges (P). Note is made of smallopaque hairs (H) near the wrist preventing penetration of NIR light.

E. The mid-tail showing caudal vertebrae (CV). Soft tissue and skinoverlying these bones can be seen as a superimposed ribbed and checkeredpattern.

BEST MODE FOR CARRYING OUT THE INVENTION I. OVERVIEW

In vertebrates, the development and integrity of the skeleton requireshydroxylapatite deposition by osteoblasts. Hydroxylapatite deposition isalso a marker of, or a participant in, processes as diverse as cancerand atherosclerosis. Prior to the present invention, sites of exposedhydroxylapatite are imaged in vivo using γ-emitting radioisotopes. Thescan times required are long, and the resultant radioscintigraphicimages suffer from relatively low resolution.

The present invention provides methods and compositions for improvedimaging of tissues which include hydroxyapatite. In particular, thepresent invention provides fluorescent bisphosphonate derivatives thatexhibits rapid and specific binding to hydroxylapatite in vitro and invivo.

Certain preferred embodiments are directed to the use of bisphosphonatescoupled to near-infrared fluorescent dyes to generate contrast agentsthat retain high affinity and rapid binding to hydroxyapatite. Relativeto visible light fluorophores, contrast agents incorporatingnear-infrared (NIR) fluorophores permit highly sensitive detection ofnew bone formation, or other hydroxyapatite-positive tissue, withminimal interference from tissue absorbance, autofluorescence, andscatter. In addition to in vitro uses, clinical applications whichcontemplates include, but are not limited to:

In vivo detection of osteoblastic metastases: The ability tonon-isotopically visualize osteoblastic metastases in a living animal,without sacrifice, should permit more rapid and efficient evaluation ofanti-tumor therapies.

Histopathological detection of vessel micro-calcification: A newparadigm in atherosclerosis research is that plaque formation has manyfeatures of osteoblastosis. The subject contrast agents permit detectionof sub-clinical vessel calcification as a way of more accuratelyassigning cardiovascular risk.

In vivo detection of vessel micro-calcification: The subject contrastagents can be used to detect vessel micro-calcification, such as for invivo NIR fluorescent imaging of vessel calcification either as anadjunct to standard catheterization procedures, or as a prognostic toolto assess cardiovascular risk.

II. EXEMPLARY COMPOUNDS

The subject compositions include contrast agents generated by theconjugation of a bisphosphonate moiety with a fluorescent moiety, suchas an organic dye or fluorescent protein. In preferred embodiments, suchcontrast agents are represented in the general formula [I], orpharmaceutically acceptable salts thereof:

wherein

D represents a fluorescent moiety (also referred to herein as a “dye”);

R represents a linking group that covalently links the dye (D) andbisphosphonate moiety;

R₁ represents H, —OH, or a halogen; and

R₂ represents, independently for each occurrence, a free electron pair,hydrogen, or a pharmaceutically acceptable counterion.

A successful bone scanning agent and bone therapy requires high andrapid uptake of the agent in bone with clearance from the blood and softtissues, such as muscle, of that part of the agent not taken up in thebone.

A. Exemplary Bisphosphonates Moieties

Bisphosphonates are synthetic analogs of naturally occurring inorganicpyrophosphates and have been used for many years in the treatment ofPaget's Disease and hypercalcemia because like inorganic pyrophosphates,bisphosphonates bind to hydroxyapatite crystals in mineralized bonematrix, inhibit the recruitment and function of osteoclasts andstimulate osteoblasts to produce an inhibitor of osteoclast formation.Bisphosphonates are resistant to metabolic and enzymatic inactivation byskeletal pyrophosphatases as they contain aphosphorous-carbon-phosphorous backbone rather than thephosphorous-oxygen-phosphorous backbone of pyrophosphates.

Bisphosphonates, pyrophosphates and bisphosphonate-like compounds,collectively referred to herein as “bisphosphonates” are those compoundsexhibiting the characteristics of compounds having aphosphate-oxygen-phosphate or phosphate-carbon-phosphate backbone whichcharacteristics comprise the ability to bind strongly calcium crystalsand affect osteoclast-mediated bone resorption. As used herein,bisphosphonates include both geminal and non-geminal bisphosphonates. Anumber of bisphosphonates are commercially available and can be adaptedfor use in the subject methods and compositions. These include, but arenot limited to:

alendronate 4-amino-1-hydroxybutylidene)bis-phosphonate

clodronate (dichloromethylene)-bis-phosphonate

EB-1053 1-hydroxy(1-pyrrolidinyl)-propylidene)bis-phosphonate

etidronate, 1-hydroiyethylylidene)-bisphosphonate

ibandronate 1-hydroxy(methylpentylamino)propylidene)bis-phosphonate

incadronate [(cycloheptylamino)-methylene]bis-phosphonate

neridronate (6-aminohydroxyhexylidene)bis-phosphonate

olpadronate ((3-dimethylamino)-1-hydroxypropylidene)bis-phosphonate

palmidronate 3-amino-1-hydroxypropylidene)bis-phosphonate)

risedronate (1-hydroxy(3-pyridinyl)-ethylidene)bis-phosphonate

tiludronate [[(4-chlorophenyl)thio)-methylene]bis-phosphonate

YH 529 (1-hydroxyimidazo-(1,2-a)pyridinylethylidene)bis-phosphonate

zoledronate 1-hydroxy-2-(1H-imidazole-1-y)ethylidene)bis-phosphonate

In certain embodiments, the bisphosphonate is selected from the groupconsisting of pyrophosphonates, thiobisphosphonates, andnitrobisphosphonates. “Nitrobisphosphonates” are compounds comprising anitrogen atom bound to two phosphonate groups. “Thiobisphosphonates” arecompounds comprising a sulfur atom bound to two phosphonate groups.

Referring to Formula I above, in certain preferred embodiments, R′ is H,—OH or —Cl, and R is an amine substituted alkyl, e.g., —(CH₂)_(L)—NH—where L is an integer from 1 to 6, which forms an amide linkage with thedye moiety.

B. Exemplary Fluorescent Moieties

As described in further detail below, fluorescent, and preferablynear-infrared detection of hydroxylapatite can be used to study skeletaldevelopment, osteoblastic nmetastasis, coronary atherosclerosis, andother human diseases.

There are a wide range of fluorescent moieties which can be used to formthe subject contrast agents. Such moieties include organic dyes as wellas fluorescent proteins.

Examples of appropriate dyes include rhodamines, indocyanines,fluoresceins, hematoporphyrins, and fluoresdamines. Examples offluorescent proteins include phytofluors (plant phytochromo apoproteins)and green fluorescent proteins. Selection of the appropriate fluorescentmoiety will also require considering such factors as (i) the ability ofthe agent to be conjugated to the bisphosphorate moiety withoutsubstantially reducing the usefulness of the fluorescent probe (e.g.retains high extinction coefficient and quantum efficiency), (ii) theresulting conjugate is water-soluble, (iii) the resulting conjugate isnon-toxic (e.g. has an LD₅₀ of 100 mg/Kg or higher, and more preferablygreater than 300 mg/K), (iv) the resulting conjugate has a sufficienthalf-life in the body (e.g., preferably grater than 10 minutes, morepreferably greater than 30 minutes).

Although any fluorescent dye may be suitable, dyes that absorb and emitradiation within the near infrared (NTR) region of the electromagneticspectrum are preferred. Bone exhibits extremely high autofluorescence inthe ultraviolet and visible ranges and tissue photon scatter in theseranges precludes imaging of deep structures. In fact, biological tissueexhibits a high photon absorbance in both the visible wavelength range(350-700 nm; secondary to hemoglobin, tissue pigments, etc.) andinfrared (>900 nm; secondary to lipids and water). However, in thewavelength range 700 nm to 900 nm (near-infrared; NIR), the absorbancespectra for all bio-molecules reach minima, thus permitting deep photonpenetration into tissue (NIR window). The benefits of using NIRfluorescent dyes as labels include.

(1) there is very low interference at the NIR wavelength of about 650 to1000 nm where only a few classes of compounds exhibit significantabsorption or fluorescence,

(2) NIR fluorescent dyes are compatible with the use inexpensivegallium-aluminum-arsenide (Ga—Al—As) semiconductor laser diodes toinduce fluorescence; and

(3) NIR detection permits flexibility in selecting siliconphotodetectors.

Compounds that possess absorbance at the lower NIR (700-850 nm), herein“near-infrared fluorescent moieties”, include phthalocyanine,indocyanine, and napthalocyanine dyes, metal complex dyes, triphenyl- ordiphenylmethanes, azo dyes, quinones, and carbocyanine dyes.

Cyanine dyes with intense absorption and emission in the near-infrared(NIR) region are particularly useful because biological tissues areoptically transparent in this region (B. C. Wilson, Optical propertiesof tissues. Encyclopedia of Human Biology, 1991, 59 587-597). Forexample, indocyanine green, which absorbs and emits in the NIR regionhas been used for monitoring cardiac output of hepatic functions, andliver blood flow (Y-L. He, H. Tanigami, H. Ueyama, T. Mashimo, and 1.Yoshiya, Measurement of blood volume using indocyanine green measuredwith pulse-spectrometry: Its reproducibility and reliability. CriticalCare Medicine, 1998, 26(8), 1446-1451; J. Caesar, S. Shaldon, L.Chiandussi, et al., The use of Indocyanine green in the measurement ofhepatic blood flow and as a test of hepatic function. Clin. Sci. 1961,21, 43-57) and its functionalized derivatives have been used toconjugate biomolecules for diagnostic purposes (R. B. Mujumdar, L. A.Ernst, S. R. Mujumdar, et al., Cyanine dye labeling reagents:Sulfoindocyaninesuccinimidyl esters. Bioconjugate Chemistry, 1993, 4(2),105-1 1 1; Linda G. Lee and Sam L. Woo. “N-Heteroaromatic ion andiminium ion substituted cyanine dyes for use as fluorescent labels”,U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al. “Light imaging contrastagents”, WO 98/48846; Jonathan Turner, et al. “Optical diagnostic agentsfor the diagnosis of neurodegenerative diseases by means of nearinfrared radiation”, WO 99/22146; Kai Licha, et al. “In-vivo diagnosticprocess by near infrared radiation”, WO 96/17628; Robert A. Snow, etal., Compounds, WO 98/48838).

In certain preferred embodiments, the fluorescent contrast agent isrepresented by the formula [II]

wherein

R₃, independently for each occurrence, is a substituted or unsubstitutedalkyl;

A and B are each, independently, 5-7 membered substituted orunsubstituted aromatic rings;

L₁- L₇ are each, independently, a substituted or unsubstituted methine,provided that one of L₁-L₇ is substituted with a linker group (R,defined above) which is attached to the bisphosphonate;

X and Y are the same or different and each is a group of the formula—O—, —S—, —CH═CH— or —C(R₄)₂—;

R₄, independently for each occurrence, is a hydrogen or substituted orunsubstituted alkyl; and

r is 0, 1 or2.

Exemplary fluorescent contrast agents of this nature are described, forexample, in PCT publication WO00/16810. In certain preferredembodiments, the fluorescent contrast agent is characterized by one ormore of the following features: (a) it is free of a carboxylic acidgroup in a molecule; (b) r is 1; (c) 4 or more sulfonic acid groups arecontained in the molecule; (d) 10 or less sulfonic acid groups arecontained in a molecule; (e) A and B are, independently, benzo ornaphtho rings; and (f) X and Y are —C(CH₃)₂—.

In certain embodiments, any two of L₁-L₇ are substituted and form aring. An example of such an embodiment is the fluorescent contrast agentrepresented by the formula [III]

wherein

X and Y are as defined above, and preferably are each —C(CH₃)₂—;

R₆, independently for each occurrence, is hydrogen or —SO₃R₇;

R₇, independently for each occurrence, is hydrogen or a pharmaceuticallyacceptable counter ion, and is preferably sodium (NA);

m is 0, 1, 2, 3, 4 or 5. and is preferably 4.

Exemplary dyes of this nature are available commercially, such as fromLI-COR, Inc. (Lincoln, Nebr.) and can be readily conjugated to abisphosphonate moiety. For instance, the subject imaging agents can bederived through the use of IRDye80, IRDye78, IRDye38, IRDye40, IRDye41,IRDye700, or IRDye800 from LI-COR, Inc. (see also U.S. Pat. No.6,027,709). These dyes include carbodiimide (CDI) active groups that canbe covalently bonded to a biphosphonate moiety.

In comparison to other fluorophore/sensitizers, the photoproperties ofsulphonated indocyanine derivatives such as IRDye78 (LiCor catalognumber 829-05932) are optimal for in vivo use. Analysis has revealedpeak absorption at 771 nm and peak emission at 796 nm for IRDye78, areasof the spectrum with the lowest possible tissue absorbance. IRDye78 hasa high extinction coefficient (150,000 M⁻¹ cm⁻¹) in aqueous medium, andΦ_(F) of 34%. Finally, IRDye78 has been specifically designed forconjugation to other biomolecules.

As described in the appended examples, one preferred contrast agent isan amide linked conjugate of pamidronate and IRDye78. Pamidronate is aprimary amine-containing bisphosphonate, which can be coupledconveniently to NIR fluorophores. Pamidronate is conjugated to anear-infrared fluorophore. In preferred embodiments, the fluorophore ise.g., preferably one whose excitation maximum is about 771 nm and has anemission maximum is 796 nm, and extinction coefficient is 150,000 M⁻¹cm⁻¹ in aqueous environments. Purification of the product (Pam78) hasbeen optimized by utilizing thin-layer chromatography. Pam78 binds to HAcrystals with rapid kinetics, is competed with unconjugated pamidronate,and has a binding capacity consistent with previously publishedbisphosphonate conjugates.

C. Exemplary Formulations

Acute, sub-acute, and chronic administration of bisphosphonates has, ingeneral, revealed little toxicity. This is generally explained by theirrapid incorporation into calcified tissue and hence their short presencein the circulation. Accordingly, a wide variety of formulations androutes of administration are expected to be available for the subjectcontrast agents.

The compounds of the present invention can be administered to amammalian host in a variety of forms adapted to the chosen route ofadministration, i.e., orally, or parentally. Parenteral administrationin this respect includes, but is not limited to, administration by thefollowing routes: intravenous, intramuscular, subcutaneous, intraocular,intrasynovial, transepithelially including transdermal, opthalmic,sublingual, and buccal; topically including opthalmic, dermal, ocular,rectal and nasal inhalation via insufflation and aerosol and rectalsystemic.

The active compound may be orally administered, for example, when aninert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsules, or it may be compressedinto tablets, or it may be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipient and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium sicarate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl, and propylparabens as apreservative, a dye and flavoring. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound maybe incorporated into sustained-release preparations andformulations. The active compound may also be administered parenterallyor intraperitoneally. Solutions of the active compound as an ester, afree base or a pharmacologically acceptable salt can be prepared inwater or other aqueous solution (e.g. water suitably mixed with asurfactant such as hydroxypropylcellulose). Dispersion can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable, use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It may be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent of dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbicacid, thimersal, and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be obtained by the use of agents delayingabsorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, typicallyfollowed by filtered sterilization. Generally, dispersions are preparedby incorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

D. Exemplary Uses

The subject compositions can be used as part of an imaging technique toanalyze bone and other tissue which may include hydroxyapatite.

An aspect of the invention is a method for evaluating a bone for itscondition and biomechanical property. In certain embodiments, thesubject contrast agents can be used as part of an in vivo imagingprotocol for determining bone matrix density. In a particularembodiment, the invention contemplates measuring bone mineral density(BMD) of a selected region of bone in a small body portion of a human orother animal

In this regard, the subject method can be used for example, to detectchanges in bone matrix volume which correspond to the condition of thebone.

Thus, the invention provides a method for diagnosing osteoporosis whichincludes the use of the subject contrast agents. In this approach, ameasure of trabecular thickness and bone perimeter, determined from boneimages can be used together to assess the condition of trabecular boneat the site of interest.

In other embodiments, the subject contrast agents and imaging materialscan be used to determine the biomechanical properties of bone in vivo,wherein the biomechanical properties are reflected in a set ofanisotropic elastic constants, bone strength, or fracture risk.

Still another embodiment of the subject method is for diagnosticdetection of bone diseases accompanied with abnormality of calciumhydroxyapatite, such as metastasis of cancer to bone, especially at theinitial stage.

This invention provides imaging methods for use in an animal, whichincludes, but is not limited to, humans and other mammals. The term“mammals” or “mammalian subject” also includes farm animals, such ascows, hogs and sheep, as well as pet or sport animals such as horses,dogs and cats.

III. EXEMPLIFICATION

As described further below, NIR fluorescent bisphosphonate derivativeswere synthesized and demonstrated to have nearly ideal optical and HAbinding properties. Using this compound, what is believed to be thefirst NIR fluorescence imaging of a complete body system, the growingskeleton, in a living animal was obtained. In the same animal, NIRfluorescence imaging was compared to ^(99m)Tc-MDP radioscintigraphy andmagnetic resonance imaging.

A. Conjugation and Purification of a Near-Infrared Fluorescent SmallMolecule Ligand with Specific Binding to Hydroxyapatite

The small molecule pamidronate (FIG. 1A, molecule I), which has specificbinding to HA, was chosen as the bisphosphonate for this study since ithas a single primary amine for conjugation and is readily available inpowder form. It also has one of the shortest spacing elements (twocarbons) between the HA binding portion of the molecule and the site ofconjugation, hence providing a stringent test for the concept of usingprimary amine-containing bisphosphonates as ligands to which bulky NIRfluorophores can be attached.

Covalent conjugation to the N-hydroxysuccinimide (NHS) ester of the NIRfluorophore IRDye78 (LI-COR, Lincoln, Nebr.; FIG. 1A, molecule II) wasaccomplished in one step as detailed in the Experimental Protocol. Theresultant product, termed Pam78 (FIG. 1A, molecule III), was purifiedusing thin-layer chromatography (TLC; FIG. 1B) and confirmed by massspectroscopy (data not shown). Due to pamidronate's extreme insolubilityin organic solvents, and IRDye78's base lability, the conjugationreaction had to be performed under near-neutral aqueous conditions.Despite this, we were able to achieve Pam78 yields of IS-21%,

B. Spectral Properties of IRDye78 and Pam78

Absorbance wavelength scans of IRDye78 (FIG. 1C, left panel) and Pam78(FIG. 1C, right panel) were performed as described in the ExperimentalProtocol. IRDye78 had a peak absorption at 771 nm, with a minor peak at708 nm (discussed below). Peak absorption of Pam78 was unchanged fromIRDye78, although the 708 nm peak was significantly more pronounced.Emission fluorescence scans (FIG. 1D, dashed lines), with excitationwavelength fixed at 771 nm, were performed. Both IRDye78 (FIG. 1D, leftpanel) and Pam78 (FIG. 1D, right panel) had a peak emission at 796 nm.Of note, excitation at the minor absorption peak of 708 nm resulted inidentical peak emission wavelengths for both IRDye78 and Pam78 (data notshown), suggesting that this minor absorption peak is an alternativeresonance structure and not an impurity in the preparation. Conjugationto pamidronate likely stabilizes this alternative resonance (compareFIG. 1C, left panel to right panel).

C. In Vitro Hydroxyapatite-Binding Properties of IRDye78 and Pam78

Bisphosphonates bind HA rapidly and with high specificity [4]. Todetermine whether pamidronate has bulk tolerance for relatively largemolecules such as IRDye78, we performed a kinetic binding assay (FIG.2A). Pam78 exhibited rapid binding to HA, with 37% of peak bindingachieved in just five minutes and asymptotic binding by 1 hour.Continued binding to HA was seen even at the longest time point (24hours) consistent with previously published results [4]. The carboxylicacid of IRDye78 (FIG. 2A) exhibited no detectable binding to HA during24 hours of incubation.

As observed previously, HA has an extremely high, and essentiallyunsaturable capacity for bisphosphonates, precluding measurement of anequilibrium binding constant [4]. However, we sought to determine if thecapacity of HA to bind pamidronate was altered by conjugation toIRDye78. FIG. 2B shows the results of such an experiment. The measuredbinding capacity, plotted as a function of applied bisphosphonateconcentration, was lower than previously published results for similar,but unconjugated, bisphosphonates [4] by a ratio exactly equal to theratio of their respective molecular weights. This suggests that bindingto HA was unaltered by conjugation, but the total capacity of bindingwas lowered by steric hindrance. Consistent with the experiment shown inFIG. 2A, IRDye78 alone exhibited no binding to HA.

Designed as pyrophosphate-mimetics, bisphosphonates have extremely highspecificity for hydroxyapatite both in vitro [4] and in vivo [20].Nevertheless, we sought to confirm that Pam78 binding to hydroxyapatitecould be competed by unlabeled pamidronate. FIG. 2C shows the results ofsuch a competition experiment in which 2.5 μM Pam78 was bound to 1.5mg/ml (1.5 mM) HA crystals in the presence of increasing concentrationsof unlabeled pamidronate. Theoretical estimates suggest that HA has amaximum binding capacity of approximately 8.4×10⁻⁴ moles pamidronate pergram HA. Hence, the HA in each 100 μl reaction would be expected to havea capacity of 126 nmol of pamidronate. As shown in FIG. 2C, Pam78 waseffectively competed by increasing concentrations of unlabeledpamidronate, with 90% inhibition seen in the presence of 80 nmol (800μM) unlabeled pamidronate.

D. In Vivo Near-Infrared Fluorescence Imaging of HA Using IRDye78 andPam78

The small animal imaging system described in the Experimental Protocolis similar to one published previously [21]. It has an adjustablecircular field of view (FOV) with a theoretical and measured resolution,respectively, of 100 μ and 200 μ at a of FOV diameter of 10 cm, and 10 μand 25 μ at a FOV diameter of 1 cm. Fluorescence excitation powerdensity of the system was 18 mW/cm², with minimal spatial variation.Zoom lens and camera were chosen for excellent transmission andsensitivity, respectively, at 800 nm.

In preliminary experiments, it was determined that body hair causessignificant NIR light scatter and essentially precludes planar NIRimaging of small animals (see also below). Hence, hairless (nu/nu) micewere used throughout this study. FIG. 3A shows a typical experimentusing the carboxylic acid of IRDye78 injected intravenously.Autofluorescence of the animal using an excitation band of 750±25 nm andemission band of 810±20 nm was negligible (FIG. 3A, FIG. 3B). Within oneminute, and appearing relatively stable for the next fifteen minutes,the entire mouse became fluorescent (FIG. 3A). Of note, any smalldefects present on the animal's skin became pronounced after IRDye78injection (FIG. 3A; discussed below). Over the ensuing six hours,IRDye78 was eliminated rapidly by the genitourinary and biliary systems(FIG. 3A). Gallbladder filling and contraction, and intestinalperistalsis were easily visible during this time (data not shown). Bysix hours, NIR fluorescence had returned to near background except inthe above sites of elimination (FIG. 3A).

Intravenous injection of Pam78 (FIG. 3B) resulted in similarly rapiddistribution throughout the body and intense NIR fluorescence of theanimal. However, as Pam78 was cleared by the genitourinary and biliarysystems, areas of exposed HA became visible. As early as 15 minutespost-injection, Pam78 uptake in the spine, ribs, paws and knees could bedetected above background, and by three hours, most bony structures werevisible (FIG. 3B). Although a 500 msec exposure time was used throughoutthis study to match the full dynamic range of the camera, acquisitiontimes as short as 50 msec resulted in excellent quality images, whichsuggests that real-time dynamic NIR fluorescence imaging will bepossible with newly introduced interlined NIR cameras.

E. Pharmacokinetics and Percentage Uptake of IRDye78 g and Pam78

Although the fluorescent signal from the entire animal could be followedover time, we sought to quantitate the rate of plasma elimination ofIRDye78 and Pam78 after intravenous injection. The data (FIG. 3C)suggest that IRDye78 achieves peak concentrations within 1 minute afterinjection and exhibits a two-phase elimination from the plasma, withearly and late half-lives of 7.2 and 24.7 minutes, respectively. Thelate in vivo half-life is consistent with the late in vivo half-livesreported for similar poly-sulphonated indocyanine NIR fluorophores [22].Pam78 also shows peak plasma concentration by 1 minute, with a morerapid clearance having early and late half-lives of 5.0 and 15.4minutes, respectively. This is not surprising given the rapid in vivobinding of Pam78 to HA simultaneous with its elimination by thegenitourinary and biliary systems.

F. Comparison of Near-Infrared Fluorescence Imaging to ^(99m)Tc-MDPRadioscintigraphy

Six hours after Pam78 injection, fluorescent background was such thatoptimal images were obtained. All of the dorsal bony structuresdelineated in FIG. 3B became better defined, and ventral imagingrevealed additional structures such as bones of the maxilla, sternum andknees.

Prior to this study, the gold standard for imaging of HA was a bone scanwith ^(99m)Tc-MDP. To compare, directly, these two imaging methods, thesame animal was re-injected with ^(99m)Tc-MDP and imaged by planarradioscintigraphy six hours later. Due to the inherent tradeoff betweenpinhole size and FOV, it was not possible to image the entire animalwith higher resolution than that shown. Also, the radioscintigraphicimage required a 30 minute integration time for 0.4 mCi of ^(99m)Tc-MDPinjected versus a 500 msec image acquisition time for NIR fluorescenceusing 2.6 nmol of Pam78.

G. Target Depth and Effects of Scatter on Signal Intensity and ImageQuality Using Near-Infrared Fluorescent Ligands

The correlation of anatomical landmarks imaged by MRI with NIR signalintensity permits the modeling of observed NIR fluorescence signal as afunction of target depth beneath the skin surface. The net NIRfluorescent signal intensities of the first seven visible spinousprocesses were plotted against their depth beneath the surface of theskin as measured by MRI. The data fit an exponential decay curve wheremeasured intensity is proportional to e^(−kd), k=−0.43/mm and d=distancefrom the skin surface to the target in millimeters (R² of fit=0.98). Thesignificance of this model is discussed below.

H. Quantitation of Pam78 Skeletal Uptake

To quantitate percentage uptake of injected dose, we first tried toisolate Pam78 directly from excised bones. Unfortunately, the harshconditions necessary for bone dissolution (6 N HCl) destroyed thefluorophore (data not shown). However, we were able to estimate skeletaluptake by measuring fluorescence intensity of the ribs in the skinlessanimal. Calibration standards matching the geometry of the ribs wereplaced next to the animal and mean fluorescence intensities of eachstandard and the caudal three ribs on each side were measured. Pam78concentration (mean±S.E.M.) was found to be 0.91±0.027 μM. Assuming abone density of 1.8 g/cm³ [23] and a skeletal weight of 11.8% bodyweight [24], 1.5 nmol (57%), out of 2.6 nmol injected, bound to theskeleton. The skeletal uptake of Pam78 compares favorably with the 52%uptake reported in rats using ^(99m)Tc-MDP [25].

I. High-Resolution in Vivo Near-Infrared Fluorescence Imaging of HA inSpecific Anatomic Sites

The small animal NIR fluorescence imaging system described above wasdesigned to include zoom capability. Six hours after Pam78 injection,the same animal shown in FIGS. 3B and 4 was used to obtainhigh-resolution images of various sites of exposed HA (FIG. 5). Althoughscatter from the skin and overlying soft tissue resulted in blurring ofsome structures, most bones of the animal could be visualized and some,like the bones of the maxilla, could be seen with extremely high clarity(discussed below).

J. Discussion of Results

This study highlights many of the principles of NIR fluorescenceimaging. Of paramount importance is the choice of fluorophore. The NIRwindow has a local minimum at approximately 800 nm, and fluorophoreswith excitation/emission wavelengths close to 800 nm should permitmaximal photon penetration into living tissue. IRDye78 and itsderivatives have an excitation wavelength of 771 nm, emission wavelengthof 796 nm, and a relatively high extinction coefficient and quantumefficiency. The NHS ester of IRDye78 permits one-step conjugation toprimary-amine-containing ligands, and products are easily purified usinglow cost TLC. Poly-sulphonated indocyanines such as IRDye78 haveextremely low toxicities, and plasma half-lives in the range of 10-30minutes result in rapid clearance of background signal. Pam78 alsoappears to be stable in vivo since even twelve hours post-injectionthere is no degradation of skeletal signal (data not shown). Thissuggests that the amide bond between pamidronate and IRDye78, and thefluorophore itself, were not hydrolyzed significantly.

Pam78 provides a convenient reagent for creating “embedded targets” in aliving animal. NIR fluorescence imaging systems are usually evaluatedusing fluorescent phantoms that seldom have relevance to in vivoimaging. Pam78, however, creates an essentially infinite number oftarget structures embedded at all depths within a living animal. Such areagent should be extremely valuable for creating the much needed nextgeneration of NIR fluorescence imaging systems.

By measuring the observed NIR fluorescence intensity as a function ofoptical path length between excitation light and target, we were able toderive the exponential decay constant of k=−0.43/mm. This model predictsthat the presence of skin (0.89 mm thick on the back of a 25 g nudemouse; AZ and JVF, unpublished observations) would reduce net NIRfluorescence signal intensity by 32%. After adjusting for exposure time,comparison of the animal with and without skin resulted in an intensityattenuation of 44%, in reasonable agreement with the model. This modelmay be useful as a frame of reference for investigators contemplatingthe use of NIR fluorescence for target detection in vivo. Of course, thepresence of skin markedly reduces the quality of the image due toscatter. Optical scatter is a major limitation to planar imaging usingNIR light. The use of an optical coupling medium and/or tomographicimaging may provide a means of minimizing this effect.

Pam78 has several immediate applications in the study of skeletaldisease. It provides high resolution imaging of HA without compromisingsensitivity and specificity (FIGS. 3B, 5). Certain structures such asthe bones of the maxilla (FIG. 5A) are seen with near-photographicclarity, which should permit detailed, non-invasive study ofmaxillofacial development. Skeletal development in higher and lowervertebrates, as well as animal models of bony disease, could be studiedwith this technology. Although the present study was conducted withhairless animals, chemical hair removal using commercially availableproducts should permit any mouse strain to be used (AZ and JVF,unpublished observations).

Pathologic skeletal processes such as osteoblastic metastases shouldalso be readily detectable using this technology. Presently, the goldstandard for imaging exposed HA is radioscintigraphy with ^(99m)Tc-MDP.To compare directly NIR fluorescence imaging with radioscintigraphy, wedeveloped a procedure by which the same animal was imaged sequentiallywith both modalities. In addition, the same animal was imaged with highfield magnetic resonance imaging so that all results could becross-correlated to the precise location of anatomical landmarks. As onemight expect, there were distinct advantages to each method. NIRfluorescence imaging was extremely rapid (500 msec or less for completeimage acquisition and processing), had high sensitivity after only 2.6nmol of injected Pam78, and offered relatively high spatial resolution(FIG. 5). However, deep structures were either poorly visualized or notvisualized at all with our experimental apparatus due to skin and softtissue attenuation and scatter. In comparison, planar radioscintigraphyrequired long integration times, and a tradeoff between FOV and spatialresolution. However, imaging of deep structures was possible since the140 keV γ-ray is highly penetrant and minimally scattered. We proposethat NIR fluorescence detection may permit the earliest genetic changesassociated with osteoblastic metastases to be studied more easily inmouse model systems since small lesions can be detected rapidly, withhigh resolution, and non-isotopically in the living animal.

The technology described in this study may also have severalnon-skeletal applications. First, the carboxylic acid of IRDye78distributes rapidly throughout the body and is able to highlight evensmall defects in the skin (FIG. 3A). It may be possible, therefore touse IRDye78 as a part of a sensitive detection system for dermatologicdisease. Indeed, a related NIR fluorophore, indocyanine green, hasalready been used in such a a manner. Second, there are several effectsof pamidronate, such as direct osteoclast inhibition and parasitetoxicity, that defy mechanistic explanation at present. Pam78 may be avaluable reagent to visualize the binding of pamidronate to osteoclastsand parasites in the hope of better defining its mechanism of action.Third, middle/inner ear anomalies caused by hydroxyapatite deposition,such as otosclerosis, could be detected with high sensitivity andspecificity by injecting Pam78 intravenously, placing a fiberscopeagainst the tympanic membrane, and directly visualizing NIRfluorescence.

Finally, perhaps one of the most exciting applications of thistechnology will be in imaging the earliest manifestations of coronarycalcification. Even ^(99m)Tc-MDP is capable of imaging aorticcalcification, but only in advanced disease and with low resolution.Intravenous or intracoronary injection of Pam78 would be expected tohighlight even microscopic HA deposits, since intimal thickness is wellwithin the range of this technology. Analogous to intravascularultrasound, a NIR fluorescence angioscope inserted into a coronaryartery should permit visualization of the earliest precursor lesions ofvascular calcification and, hence, permit intervention before aconventional plaque forms. Indeed, if it is determined that the yield oftriplet formation of Pam78 is sufficient, it may be possible to performboth detection (via fluorescence angioscopy) and treatment (viaphotodynamic therapy) of sub-clinical atherosclerotic lesions with thissingle compound.

K. Experimental Protocol

Reagents. Pamidronate disodium was a generous gift from InterchemCorporation (Paramus, N.J.). The NHS ester of IRDye78™ was purchasedfrom LI-COR (Lincoln, Nebr.) and stored under nitrogen at −80° C. untiluse. Immediately before conjugation, it was resuspended in DMSO to 22.8mM. The carboxylic acid of IRDye78 was a generous gift from LI-COR(Lincoln, Nebr.). Hydroxyapatite (CAS# 1306-06-5; MW 1004.6) waspurchased from Calbiochem (La Jolla, Calif.; Catalog#391947).Fluoromount-G was purchased from Southern Biotechnology Associates, Inc.(Birmingham, Ala.). All other chemicals were purchased from Sigma (St.Louis, Mo.). H₂O used in this study was purified to 18 MΩ on a Milli-Qapparatus (Millipore, Bedford, Mass.).

Conjugation and Purification of Pam78. All steps were performed underreduced light conditions. 42.5 mM pamidronate in H₂O was adjusted with0.1 N NaOH to pH 8.5. In a 1 ml reaction were mixed 10 mM pamidronateand 5.5 mM IRDye78 NHS ester, with the remainder of the volume H₂O. Thereaction proceeded 18 hours at room temperature with constant motion.Reaction components were separated on a 60 Å pore size normal phasethin-layer chromatography plate (Whatman LK6DF), without spotting on thepreabsorbent layer. Mobile phase was 65% acetonitrile and 35% H₂O.IRDye78 and its products are bright green and are followed easily duringpurification. For analytic preparations, unconjugated pamidronate wasvisualized by spraying the TLC plate with 0.25% ninhydrin in acetone andheating. The desired product band was scraped with a razor blade, andthe silica fragments were placed in a 15 ml polypropylene tube. Afterelution with 20% acetonitrile and 80% H₂O, silica fragments were removedby passage through a 0.2 μ Durapore PVDF Ultrafree-MC membrane filter(Model UFC3-0GV-25, Millipore, Bedford, Mass.). After lyophilization,the final product was resuspended in H₂O, re-filtered as above, andstored in the dark at −80° C. until use. Concentration of the finalproduct and yield were calculated by measuring the absorbance at 771 nm,using an extinction coefficient of ε=150,000 M⁻¹ cm⁻¹.

Spectral Measurements. Absorbance measurements and scans were performedon a Model DU-600 spectrophotometer (Beckman, Fullerton, Calif.) using 1μM of each compound in 20 mM N-ethylmorpholine (NEM), pH 7.4 and 150 mMKCl (NK buffer). Fluorescence measurements, and excitation/emissionscans were performed on a SPECTRAmax Gemini XS microplate reader(Molecular Devices, Sunnyvale, Calif.) using Model 655076 black 96-wellplates (Greiner, Lake Mart Fla.), and 500 nM of each compound in NKbuffer. Curves were analyzed using SOFTmax PRO software (MolecularDevices, Sunnyvale, Calif.) and plotted using Prism3 for the Macintosh(GraphPad Software, Inc., San Diego, Calif.).

In Vitro Hydroxyapatite Binding Experiments. For kinetic measurements ofbinding to HA, 5 μM Pam78 or the carboxylic acid of IRDye78 was addedfrom a 20X stock solution to 100 μl NK buffer containing 1.5 mg/ml (1.5mM) HA. Suspensions were continuously vortexed at 37° C. for the timeindicated. Bound and unbound material was separated using a 0.2 μmfilter in a 96-well format (Model MAGVN2210, Millipore, Bedford, Mass.),and the concentration of material in the filtrate was measured using theSPECTRAmax Gemini XS fluorescence microplate reader set for anexcitation wavelength of 771 nm and an emission wavelength of 796 nm.Bound material was calculated by subtracting unbound compound from thetotal added to each reaction.

For measurement of steady-state binding to HA, the indicatedconcentration of Pam78 or the carboxylic acid of IRDye78 was added froma 20X stock solution to 100 μl NK buffer containing 1.5 mg/ml (1.5 mM)HA. Suspensions were continuously vortexed at 37° C. for three hours andprocessed as described for kinetic experiments.

For competition experiments, free pamidronate was pre-incubated in 100μl NK buffer with 1.5 mg/ml (1.5 mM) HA for five minutes at roomtemperature with continuous vortexing. Pam78 was then added from a 20Xstock solution to a final concentration of 2.5 μM. Incubation at 37° C.with continuous vortexing continued for three hours prior to filtrationand measurement as described above.

For NIR fluorescence imaging of HA crystals, 1 μM of Pam78 or thecarboxylic acid of IRDye78 in 100 μl NK buffer was incubated with 1.5mg/ml (1.5 mM) HA for fifteen minutes at room temperature withcontinuous vortexing. Crystals were washed 3× with NK buffer,resuspended in Fluoromount-G, and mounted on glass slides.

Near-Infrared Fluorescence Microscopy. NIR fluorescence images wereacquired on a Nikon Eclipse TE-300 epi-fluorescence microscope equippedwith a 100W Mercury light source, NIR compatible optics, and aNIR-compatible 10X PlanFluor objective lens (Model 93171, Nikon,Melville, N.Y.). A custom IRDye78 filter set (Chroma TechnologyCorporation, Brattleboro, Vt.) was comprised of a 750±25 nm excitationfilter, a 785 nm dichroic mirror, and an 810±20 nm emission filter.Images were acquired on a Photometrics Sensys CCD Camera (Model 1401,Roper Scientific, Tucson, Ariz.) with heat filter removed. Imageacquisition and analysis was performed using IPLab software(Scanalytics, Fairfax, Va.).

Small Animal In Vivo Near-Infrared Fluorescence Imaging System. Thesystem was constructed on an anti-vibration table (TechnicalManufacturing Corporation, Peabody, Mass.) and consisted of two 150 Whalogen light sources (Model PL-900, Dolan-Jenner, Lawrence, Mass.) towhich were mounted light guides (Model BXS-236) and 750±25 nm excitationfilters (Chroma Technology Corporation, Brattleboro, Vt.). Emitted lightpassed through an 810±20 nm emission filter (Chroma TechnologyCorporation, Brattleboro, Vt.) and was collected using a Macrovideo zoomlens with detachable close-up lens (Optem Avimo Precision Instruments,Fairport, N.Y.), into the above Photometrics Sensys CCD Camera. Imageacquisition was controlled using IPLab software and typically consistedof a 250 to 500 msec acquisition time using a camera gain of 2.Excitation power density was measured using a calibrated thermopiledetector (Model 2M) and 1000 V/V pre-amplifier (Model 1010; both fromLaser Components, Santa Rosa, Calif.).

Compound Injection and Measurement of Serum Concentration. Hairless 7week old male nu/nu mice (Charles River Laboratories, Wilmington, Mass.;average weight 25 g) were anesthetized with 50 mg/kg i.p. pentobarbital.The lateral tail vein was visualized using a flashlight and 2.6 nmol ofcompound resuspended in 80 μl of phosphate buffered saline (PBS) wasinjected using a bent, 30 gauge, ½″ needle. For measurement of plasmaconcentration, the tip of the tail of an anesthetized animal was cutwith a razor blade, and 75 μl of whole blood was milked, and collectedwith a capillary tube. After clotting and removal of cells bycentrifugation, serum was diluted 1:10 in 50 mM Hepes, pH 7.4 and awavelength scan from 400 nm to 850 nm was performed. Serum concentrationwas determined by peak height at 771 nm after subtraction of background(pre-injection serum), and correction for hemoglobin absorbance.

Magnetic Resonance Imaging (MRI). Animals were anesthetized with 50mg/kg i.p. pentobarbital, placed in a custom low-pass birdcage coil (10cm length, 6 cm diameter), and imaged with a 3T whole body scanner(General Electric Medical Systems, Waukesha, Wis.). Images were acquiredwith an 8 cm FOV using a 3D fast gradient echo sequence. Otherparameters included a 256×256 pixel matrix size, slice thickness of 0.7mm, TE=2.6 msec, TR=10.2 msec, and flip angle=15°.

Planar ^(99m)Techetium-MDP Radioscintigraphy. Anesthetized animals wereinjected with 0.4 mCi ^(99m)Tc-MDP and imaged six hours later. Dorsalimages in a 512×512 pixel format were integrated for a total of 30minutes via a custom 1 mm tantalum pinhole mounted on a Forte gammacamera (ADAC Laboratories, Milpitas, Calif.).

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1. A contrast agent represented in the general formula [I] orpharmaceutically acceptable salts thereof:

wherein D represents a fluorescent moiety; R represents a linlking groupthat covalently links the dye (D) and bisphosphonate moiety; R₁represents H, —OH, or a halogen; and R₂ represents, independently foreach occurrence, a free electron pair, hydrogen, or a pharmaceuticallyacceptable counterion.
 2. The agent of claim 1, wherein R is an aminesubstituted lower-alkyl which forms an amide bond with a pendant groupof the fluorescent moiety.
 3. The agent of claim 2, wherein R is—(CH₂)_(L)—NH—, where L is an integer from 1 to
 6. 4. The agent of claim1, 2 or 3 wherein R′ is —OH or —Cl.
 5. The agent of claim 1, 2 or 3,wherein D is a near-infrared fluorescent moiety.
 6. The agent of claim5, wherein D is a near-infrared fluorescent dye.
 7. The agent of claim5, wherein D is a polysulfonated indocyanine dye.
 8. The agent of claim1, wherein the fluorescent moiety is represented by the formula [II]:

wherein L₁-L₇ are each, independently, a substituted or unsubstitutedmethine, provided that one of L₁-L₇ is substituted with the linker groupR which is attached to the bisphosphonate, R₃, independently for eachoccurrence, is a substituted or unsubstituted alkyl; A and B are each,independently, 5-7 membered substituted or unsubstituted aromatic rings;X and Y are the same or different and each is a group of the formula—O—, —S—, —CH═CH— or —C(R₄)₂—; R₄, independently for each occurrence, isa hydrogen or substituted or unsubstituted lower alkyl; and r is 0, 1 or2.
 9. The agent of claim 8, wherein the fluorescent moiety has one ormore of the following features: (a) is free of a carboxylic acid groupin a molecule; (b) r is 1; (c) includes 4 or more sulfonic acid groups;(d) includes 10 or less sulfonic acid groups; (e) A and B are,independently, benzo or naphtho rings; and (f) X and Y are —C(CH₃)₂—.10. The agent of claim 1, wherein the fluorescent moiety is representedby the formula [III]

wherein X and Y are the same or different and each is a group of theformula —O—, —S—, —CH═CH— or —C(R₄)₂—; R₄, independently for eachoccurrence, is a hydrogen or substituted or unsubstituted lower alkyl;and R₆, independently for each occurrence, is hydrogen or —SO₃R₇; R₇,independently for each occurrence, is hydrogen or a pharmaceuticallyacceptable counter ion; m is 0, 1, 2, 3, 4 or
 5. 11. The agent of any ofthe preceding claims, wherein the near-infrared fluorescent moiety hasan extinction coefficient of at least 100,000 M⁻¹ cm⁻¹ in aqueousmediums.
 12. The agent of any of the preceding claims, wherein thenear-infrared fluorescent moiety has a quantum efficiency, Φ_(F), of atleast 25%.
 13. The agent of any of the preceding claims, having an LD₅₀of 100 mg/Kg or greater humans.
 14. The agent of any of the precedingclaims, having a half-life in the human body of at least 10 minutes. 15.A contrast agent comprising a bisphosphonate covalently linked to anear-infrared fluorescent moiety, wherein the contrast agent (a) has anextinction coefficient of at least 100,000 M⁻¹ cm⁻¹ in aqueous medium,(b) has an LD₅₀ of at least 100 mg/Kg in humans, and (c) has a half-lifein the human body of at least 10 minutes.
 16. The contrast agent ofclaim 15, wherein the bisphosphonate is selected from the groupconsisting of alendronate, clodronate. EB-1053, etidronate, ibandronate,incadronate, neridronate, olpadronate, phosphonate, palmidronate,risedronate, tiludronate, YH 529 and zoledronate.
 17. A method formanufacturing a composition of for in vivo imaging comprisingformulating a contrast agent of any of the preceding claims in apharmaceutically acceptable excipient.
 18. A kit for in vivo imagingcomprising a contrast agent of any of the preceding claims inassociation with instructions for administering the contrast agent to apatient.
 19. A method for in vivo imaging of tissue with exposedhydroxyapatite, comprising, (i) administering to the animal a contrastagent of any of claims 1-16 in an amount sufficient to renderhydroxyapatite-containing tissue detectable by a fluorescent detector;(ii) obtaining a fluorescent image of the animal, or at least a portionthereof, at a wave length(s) which detects the contrast agent; (iii)constructing an image of the animal including the pattern ofdistribution of the contrast agent.
 20. The method of claim 19, forevaluating a bone for its condition and/or biomechanical property. 21.The method of claim 20, for determining bone matrix density.
 22. Themethod of claim 20, for detecting changes in bone matrix volume.
 23. Themethod of claim 20, as part of a protocol for diagnosing osteoporosis.24. The method of claim 20, for determining at least one of anisotropicelastic constants, bone strength, or fracture risk.
 25. The method ofclaim 19, for diagnostic detection of bone diseases accompanied withabnormality of calcium hydroxyapatite.
 26. The method of claim 25, fordetecting the presence of osteoblastic metastase.
 27. The method ofclaim 19, for detecting of vessel micro-calcification.
 28. The method ofany of claims 19-27, wherein the animal is a human.